Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids

Abstract

In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after periodate oxidation and coupling by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have obtained evidence that oxidative cleavage occurs specifically at the C4 -C5 bond of core N-acetylgalactosaminitol. Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin-layer chromatography and can be sensitively analysed by liquid-secondary-ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the Nacetylgalactosamitol. These conclusions have been reached from detailed studies of the neoglycolipid derivatives of several oligosaccharides (di-to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols.The microscale structural profiling of N-linked chains of glycoproteins using hydrazinolysis, Bio-Gel P4 chromatography and specific enzyme digestion is well documented [l]. However, methods for microscale sequencing of 0-linked chains of mucin-type glycoproteins have not been developed. These chains are difficult to purify and analyse because of considerable heterogeneity in their backbone and peripheral regions and the occurrence of several types of branching at the core region N-acetylgalactosamine [2, 31. In the course of characterising neoglycolipid products [4] derived from mucin oligosaccharide alditols after mild periodate oxidation and conjugation by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have observed that oligosaccharides branched at the core N-acetylgalactosaminitol each give two derivatives. We describe here evidence that the mild periodate oxidation step specifically cleaves the C4 -C5 bond of N-acetylgalactosaminitol disubstituted at C3 and C6 or monosubstituted at C3. Reductive amination of the resulting aldehyde groups gives derivatives characteristic of each branch which can be analysed with high sensitivity by thin-layer chromatography/liquid-secondary-ion mass spectrometry. Studies of a series of oligosaccharide fractions derived from human meconium glycopeptides [3,5,6] show that this approach has considerable potential for deriving sequence

Neutral oligosaccharides of bovine submaxillary mucin

“…Fragment ions in the LSIMS permethylated spectrum, mlz 2601228 (HexNAc), mlz 4641432 (Hex/HexNAc) and rn/z 6381606 (dHexlHexlHexNAc) were in agreement with these assignments. Subsequent microscale sequence analysis based on TLC-MS of neoglycolipid derivatives [21] confirmed the presence of the major component with Fuc-Hex-HexNAc on the 6 arm of GalNAcol and HexNAc on the 3 arm (data not shown).…”

Human Trefoil Factor 2 Is a Lectin That Binds α-GlcNAc-capped Mucin Glycans with Antibiotic Activity against Helicobacter pylori

“…Glycan alditols (10 g) were converted to neoglycolipids after mild periodate oxidation by reductive amination to L-1,2-dipalminoyl-sn-glycero-3-phosphoethanolamine according to a previously published protocol for microscale applications (29). Neoglycolipids were eluted from silica gel with chloroform/methanol/water (10:10:1).…”

Thin Layer Chromatography Coupled with Surface-Enhanced Raman Scattering as a Facile Method for On-Site Quantitative Monitoring of Chemical Reactions

“…(i) because of the limited specificity and sensitivity provision capacity of TLC, spectroscopic routines like nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are needed to further characterize reaction products after the extraction of the chemicals from the TLC plate, 3,4 which is time-consuming and laborintensive; (ii) some chemicals like alkanes are fluorescence-insensitive, and cannot be detected under an ultraviolet (UV) lamp, thus prohibiting their monitoring; and (iii) since the separation on TLC is mainly based on the chemicals' different affinity with the stationary and mobile phase, chemicals with similar structure or polarity are difficult to separate completely. To overcome these limits, various technologies, such as infrared (IR), Raman, and mass spectrometry have been explored with TLC for compound specific information.…”